In this study, confocal laser scanning microscopy was used to examine the spatial and temporal dynamics of microbial colonization of Populus roots using microbial isolates expressing GFP. Standard molecular methods were used to introduce GFP into microbial isolates. Atomic force microscopy was also used to characterize the morphology, surface characteristics, and dynamics of biofilm formation of selected microbes isolated from the Populus rhizosphere. Gamma-proteobacteria isolate YR343 was observed to attach to Poplar roots after approximately 5 hours co-culture. The cells were observed to grow and form colonies on the surface of the root. Microbial isolate GM30 was examined during biofilm formation using atomic force microscopy. Expression of pili over the time course of biofilm formation and distinct morphotypes were revealed by the AFM image analysis. These colonization studies provide direct evidence that microbes collected from the rhizosphere directly associate with Populus roots. The dynamics of colonization were observed in real time using GFP-expressing microbes.